In the mammalian ovary, a follicle consists of a single oocyte surrounded by a layer of granulosa cells, a basement membrane and a third cell type known as theca cells. Folliculogenesis, or growth of the follicle leading to ovulation, has two stages. The first stage is the growth of preantral follicles in which the granulosa cells proliferate extensively. The second stage is the growth of antral follicles in which a fluid-filled space (the antrum) forms in the granulosa cell layer and then expands in size. Growth of antral follicles is accomplished by an increase in the size of the antrum, which is composed primarily of water. Molecules known to move through tissues by passing between cells (paracellular transport) or through cells (transcellular transport). Recently, it was discovered that a family of proteinaceous channels termed aquaporins facilitates transcellular transport of water in a number of tissues, including the testis. Preliminary data indicate that ovarian follicles in the process of developing an antrum express mRNA and protein for aquaporins-8 and -9. In preovulatory follicles (with fully mature antra), aquaporin-8 mRNA is no longer expressed, although aquaporin-9 mRNA remains constitutive. Functional assays have shown that granulosa cell permeability inhibited by HgCl2 (a characteristic of aquaporin-mediated water movement) and altered with changing aquaporin-8 mRNA levels. The results have lead to the hypothesis that aquaporins mediate water movement into the developing antrum. These experiments will investigate the role of aquaporins in antrum formation during folliculogenesis in vivo by 1) localizing the pattern of their expression in adult rats using in situ hybridization and immunocytochemistry; 2) evaluating the contribution of transcellular and paracellular transport routes to follicular water permeability and 3) growing antral follicles in the presence of aquaporin inhibitors. Together these studies will provide a novel insight into the mechanism of ovarian function and provide critical information as to the importance of these molecules in preparing a follicle for ovulation.